The epithelial membranes of the body, including the skin and the various mucosal membranes, have two layers. The surface layer or epithelial layer is mostly made of a type of epithelial cells called keratinocytes, while the underlying or dermis layer is a connective tissue which is fibrous and tightly attaches the keratinocytes to the sub-dermal layers.
An active area of research in the past several decades has been the development of human skin equivalents, which have a similar anatomical and biochemical make-up to whole skin, i.e., epidermis and dermis, and can be used to successfully treat trauma patients. The resulting skin equivalents can be applied to the trauma patients with relative ease.
A similar type of equivalent for the mucosal membranes of the body would be useful for reconstruction after trauma, surgical resection or preprosthetic surgery. The use of skin in procedures involving the mucosal membranes, however, presents several disadvantages, for example, a different pattern of keratinization between the keratinocytes of the skin and mucosal membranes. Thus, for this and other reasons, the techniques developed for skin equivalents are not readily transferable to the development of mucosal composites.
The development of mucosal equivalents has focused on the oral mucosa because of the ease of access and numerous procedures performed in the oral cavity. Previously, oral mucosa substitutes have involved split-thickness skin grafts or palatal or buccal oral mucosal grafts, both of which require at least a second surgical procedure. These procedures are unsatisfactory, though, because of the presence of adnexal skin structures, limited supply of mucosal grafts available and the uneven texture that results. Fabrication of cultured sheets of epithelial cells has been attempted in order to remedy these drawbacks. Known fabrication techniques, however, are also unsatisfactory because they rely on a feeder layer composed of irradiated mouse fibroblasts, thus risking introduction of murine DNA into proliferating human cells. The use of cultured sheets of epithelial cells has produced unsatisfactory results because they are fragile, difficult to handle, and tend to contract in size upon implantation. The addition of a dermal matrix of collagen gel has not improved fragility or ease of handling the cultured sheets of epithelial cells especially when used in the oral cavity which is laden with numerous collagenolytic enzymes. In addition, these cultured cells lack the rigidity necessary for easy transfer from the culture site to the site of use.
In addition, recent revelations that the new variant of Creutzfeldt-Jakob Disease (CJD) (colloquially “mad cow disease”) is associated with meat and products which may utilize cattle brains has made it desirable to eliminate such products from all aspects of human medical treatments. This particularly pertinent in the cell culturing area because Fetal Bovine Serum (FBS) and Bovine Pituitary Extract (BPE) are common aspects of most protocols for the growth of cells. Indeed, the use of a defined culture medium, which eliminates the use of FBS and BPE from cell growth protocols, would be advantageous given the inherent unreliability of the contents of such FBS and BPE with respect to xenogeneic agents.
Moreover, the clinical usefulness of proteins and other therapeutic agents for the treatment of human disease is now well established. In addition to classic protein and peptide vaccines, these proteins may include, among others, cytokines, cytokine inhibitors or genetically engineered antibodies. Despite some clinical success, the systemic delivery of proteins continues to be problematic. Currently parenteral infusion or injection (intravenous, intra-muscular or subcutaneous) is the common route of administration. The short half-life of proteins requires frequent administration to consistently ensure the bioavailability of the injected proteins. The frequent administration also limits clinical usefulness of proteins.
The delivery of medicaments across the mucosal membranes is generally known. The delivery of proteins and other therapeutic agents transmucosally is an attractive alternative to known delivery routes for several reasons. Mucosal membranes have a high degree of vascularity. They are easily accessible for obtaining biopsies, re-transplantation and monitoring. The mucosal membranes are continually regenerated and their biology is well understood both at the cellular and molecular levels. Furthermore, the keratinocytes of the mucosal membranes are well suited for the delivery of gene products because these cells synthesize and export a wide variety of gene products to the extracellular space.
In order to have cells secrete a medicament, the medicament or medicament-producing agent must be introduced into the host cell or tissue. One method of introduction is known as transfection when a nucleic acid is introduced into the host cells or tissue. Various methods are known which introduce biological agents into host cells or otherwise transfect a host cell but generally fall into either viral or non-viral methods. Viral methods of introduction are less favored due to the inherently uncertainty that surrounds the safety of using viruses, especially with an eye toward the treatment of humans. The advantage of viral introduction methods is that the methodologies are well understood and highly effective at delivery of the introduced biological agent.
Thus, a need remains for compositions and method that enhance graft availability and suitability, while minimizing complication and risk, and that also enhance the testing, delivery and efficacy of therapeutic agents or methods.